Programmable nuclease-peptidase compositions

Abstract

Described in certain example embodiments herein are programmable nuclease-peptidase compositions, systems, and methods for the manipulation of nucleic acids and/or polypeptides. In some embodiments, the programmable nuclease-peptidase composition comprises a repeat-associated mysterious protein (RAMP) polypeptide; a guide molecule capable of forming a RAMP-guide molecule complex with the RAMP polypeptide and directing sequence specific binding of the complex to a target polynucleotide; and a peptidase capable of binding to the RAMP polypeptide, the guide molecule, or further complexing with the RAMP-guide molecule complex, wherein binding of the RAMP-guide molecule complex to the target polynucleotide initiates binding and/or interaction of the peptidase with a target polypeptide.

1 . A programmable nuclease-peptidase composition comprising:
 a repeat-associated mysterious protein (RAMP) polypeptide;   a guide molecule capable of forming a RAMP-guide molecule complex with the RAMP polypeptide and directing sequence specific binding of the complex to a target polynucleotide; and   a peptidase capable of binding to the RAMP polypeptide, the guide molecule, or further complexing with the RAMP-guide molecule complex, wherein binding of the RAMP-guide molecule complex to the target polynucleotide initiates binding and/or interaction of the peptidase with a target polypeptide.   
     
     
         2 . The programmable nuclease-peptidase composition of  claim 1 , wherein target polypeptide interaction and/or binding occurs at, or in effective proximity to, a peptidase recognition motif in the target polypeptide. 
     
     
         3 . The programmable nuclease-peptidase composition of  claim 2 , wherein the peptidase recognition motif comprises or consists of SEQ ID NO: 3 or a sequence therein, optionally MKKD (SEQ ID NO: 25). 
     
     
         4 . The programmable nuclease-peptidase composition of  claim 1 , wherein the peptidase is a TPR-CHAT peptidase. 
     
     
         5 . The programmable nuclease-peptidase composition of  claim 1 , wherein (a) the peptidase is a TPR-CHAT peptidase and wherein the TPR-CHAT peptidase is derived from  Desulfonema ishimotonii , or a homolog, ortholog, or variant thereof; (b) the RAMP polypeptide is derived from  Desulfonema ishimotonii , or a homolog, ortholog or variant thereof; or both (a) and (b). 
     
     
         6 . (canceled) 
     
     
         7 . The programmable nuclease-peptidase composition of  claim 5 , wherein (a) the RAMP polypeptide comprises a Cas11 domain and multiple Cas7 domains or (b) the RAMP polypeptide is a Type III-E Cas polypeptide. 
     
     
         8 . The programmable nuclease-peptidase composition of  claim 7 , wherein the RAMP polypeptide comprises a Cas11 domain and multiple Cas 7 domains and further comprises a Csm3, Csm4, or Csm6 domain. 
     
     
         9 . (canceled) 
     
     
         10 . The programmable nuclease-peptidase composition of  claim 1 , wherein the target polypeptide comprises, consists of, or is coupled to an effector, wherein the effector is optionally
 a. a reporter polypeptide;   b. a signal amplification polypeptide;   c. an engineered prodrug;   d. a cargo polypeptide; or   e. a pathogenic polypeptide.   
     
     
         11 . The programmable nuclease-peptidase composition of  claim 1 , wherein the target polypeptide is a disease-associated polypeptide, optionally wherein the disease-associated polypeptide is associated with a neurodegenerative disease, a metabolic disease, an autoimmune disease, or a cancer. 
     
     
         12 . (canceled) 
     
     
         13 . A polynucleotide encoding a programmable nuclease-peptidase composition or component thereof as in  claim 1 , optionally further comprising one or more regulatory elements and wherein the polynucleotide encoding a programmable nuclease-peptidase composition or component thereof is operatively coupled to one or more of the one or more regulatory elements. 
     
     
         14 . (canceled) 
     
     
         15 . A vector or vector system comprising one or more polynucleotides according to  claim 13 , optionally wherein the vector or vector system is a viral vector or vector system, optionally an adeno-associated virus vector or vector system. 
     
     
         16 . (canceled) 
     
     
         17 . A cell or cell population comprising a programmable nuclease-peptidase composition of  claim 1 . 
     
     
         18 . A pharmaceutical formulation comprising:
 (a) a programmable nuclease-peptidase composition or component thereof as in  claim 1 ;   (b) a polynucleotide encoding (a);   (c) a vector or vector system comprising one or more polynucleotides of (b);   (d) a cell or cell population comprising (a), (b), (c), or any combination thereof; or   (e) any combination of (a)-(d);   and   a pharmaceutically acceptable carrier.   
     
     
         19 . A method of modifying a polypeptide comprising:
 introducing into a sample having one or more target polynucleotides and target polypeptides, the programmable nuclease-peptidase compositions of  claim 1 ; and   activating the peptidase via sequence specific binding of the complex to the one or more target polynucleotides such that the peptidase then binds or interacts with the one or more target polypeptides resulting in modification of the one or more target polypeptides.   
     
     
         20 . The method of  claim 19 , wherein (a) the target polypeptide modification is cleavage of the target polypeptide; (b) introducing into the sample comprises in vitro, ex vivo, or in vivo delivery of the programable nuclease-peptidase composition into a cell or cell population; or (c) both (a) and (b). 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 20 , wherein
 (a) the one or more target polypeptides are proenzymes and the modification results in conversion of the proenzyme into an active enzyme;   (b) modification of the one or more target polypeptides results in activation or deactivation of one or more cell-signaling proteins;   (c) wherein the one or more target polynucleotides are a specific transcript or set of transcripts and wherein modification of the one or more target polypeptides triggers cell death upon activating the peptidase in response to binding of the nuclease-peptidase to the specific transcript or set of transcripts; or   (d) any combination of (a)-(c).   
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 22 , wherein the guide molecule is configured to detect one or more mutations in the specific transcript or set of transcripts. 
     
     
         26 . A detection composition comprising:
 (i) a RAMP polypeptide;   (ii) a guide molecule capable of forming a RAMP-guide molecule complex with the RAMP polypeptide and directing sequence-specific binding of the complex to a target polynucleotide;   (iii) a peptidase capable of binding the RAMP polypeptide, the guide molecule, or further complexing with the RAMP-guide complex; and   (iv) a detection construct,   wherein binding of the RAMP-guide molecule complex to the target polynucleotide initiates peptidase mediated modification of the detection construct resulting in generation of a detectable signal.   
     
     
         27 . The composition of  claim 26 , wherein the detection construct comprises a peptidase recognition motif recognized by the peptidase. 
     
     
         28 . The composition of  claim 27 , wherein the peptidase recognition motif comprises or consists of SEQ ID NO: 3 or a sequence therein, optionally MKKD (SEQ ID NO: 25). 
     
     
         29 . The composition of  claim 26 , wherein the peptidase is a TPR-CHAT peptidase. 
     
     
         30 . The composition of  claim 29 , wherein (a) the peptidase is a TPR-CHAT peptidase and wherein the TPR-CHAT peptidase is derived from  Desulfonema ishimotonii  or a homolog, ortholog, or variant thereof; (b) wherein the RAMP polypeptide is derived from  Desulfonema ishimotonii , or a homolog, ortholog or variant thereof; or both (a) and (b). 
     
     
         31 . (canceled) 
     
     
         32 . The composition of claim  31 , wherein (a) the RAMP polypeptide comprises a Cas11 domain and multiple Cas7 domains or (b) the RAMP polypeptide is a Type III-E Cas polypeptide. 
     
     
         33 . The composition of  claim 32 , wherein the RAMP polypeptide comprises a Cas11 domain and multiple Cas 7 domains and further comprises a Csm3, Csm4, or Csm6 domain. 
     
     
         34 . (canceled) 
     
     
         35 . The composition of  claim 26 , wherein the detection construct comprises a polypeptide comprising a protease recognition motif recognized by the peptidase, optionally wherein the polypeptide is a fluorescent protein protease reporter. 
     
     
         36 . (canceled) 
     
     
         37 . A polynucleotide encoding one or more elements (i)-(iv) of the detection composition of  claim 26 . 
     
     
         38 . A vector system comprising one or more vectors encoding one or more of elements (i)-(iv) of the detection composition of  claim 26 . 
     
     
         39 . An engineered cell modified to express elements (i) and (iii), of the detection composition of  claim 26 . 
     
     
         40 . The engineered cell of  claim 39 , wherein the engineered cell is (a) further modified to express element (iv); (b) further modified to express element (ii); or (c) both (a) and (b). 
     
     
         41 . (canceled) 
     
     
         42 . A method for screening cell perturbations comprising;
 introducing a perturbation to a cell population comprising engineered cells of  claim 39 , along with any elements of the detection composition not already expressed by the engineered cells, and wherein the guide molecules are configured to detect one or more target transcripts associated with a specific cell type or cell state;   activating the peptidase via binding of the complex to one or more target polynucleotides such that the detection construct is modified by the activated peptidase to produce a detectable product and/or signal;   detecting an ability of the perturbation to modify expression of the one or more target transcripts by measuring a change in the detectable product and/or signal relative to a control.   
     
     
         43 . A method of detecting target polynucleotides in samples comprising:
 combining a sample or a component thereof with the detection composition as in  claim 26 ; and   activating the peptidase via binding of the complex to one or more target polynucleotides such that the detection construct is modified by the activated peptidase such that a detectable product and/or signal is produced, thereby detecting the target polynucleotide in the sample.   
     
     
         44 . The method of  claim 43 , further comprising amplifying and/or enriching the target polynucleotide, optionally wherein activating the peptidase further results in activation or generation of one or more signal amplification molecules. 
     
     
         45 . (canceled) 
     
     
         46 . A method of labeling cells comprising:
 introducing the detection composition as in  claim 26  into a population of cells, wherein the guide molecule is configured to detect one or more target transcripts associated with a particular cell type or cell state; and   activating the peptidase via binding of the complex to the one or more target transcripts such that the detection construct is modified by the activated peptidase such that a detectable product and/or signal is generated, thereby labeling cells within the cell population expressing the one or more target transcripts.   
     
     
         47 . The method of  claim 46 , wherein labeled cells are further sorted or isolated based on production of the detectable product and/or signal. 
     
     
         48 . A method of in vivo effector activation or delivery comprising:
 introducing a programmable nuclease system of  claim 1  into a cell comprising a substrate of the peptidase, wherein the substrate of the peptidase is optionally tethered to a cellular structure and wherein the substrate the peptidase is coupled to an effector.   
     
     
         49 . The method of  claim 48 , wherein the effector
 a. is capable of producing a detectable signal when activated   b. is a therapeutic molecule or prodrug;   c. is a genetic modifying molecule;   d. or any combination thereof.   
     
     
         50 . The method of  claim 48 , wherein the effector is inactive when coupled to an uncleaved substrate or wherein the effector is inactive when coupled to a cleaved substrate portion. 
     
     
         51 . (canceled) 
     
     
         52 . The method of  claim 48 , further comprising cleaving the substrate by the peptidase in response to a target RNA and activation of the peptidase of the programmable nuclease system, optionally wherein the target RNA is endogenous to the cell or is exogenous to the cell. 
     
     
         53 . (canceled) 
     
     
         54 . The method of  claim 48 , wherein the substrate is tethered to a cell membrane or a nuclear membrane.